The goal of this proposal is to understand the genesis of dense (dehydrated) sickle red blood cells (RBC). Dense cells are known to make an important contribution to the pathophysiology of sickle cell disease, but a number of issues remain unresolved. In particular, it is not clear why some sickle RBC become dense soon after emerging from the bone marrow, while others become dense slowly or perhaps not at all. Hb F appears to protect cells from becoming dense quickly, and the emergence of Hb F-augmenting therapies makes it important to understand the relationship between Hb F and dense cell formation. The specific aims of this research are (1) to determine the rate of formation of dense cells in vivo, particularly those which are on the "fast track" toward becoming dense quickly, (2) to evaluate the cellular factors, e.g. Hb F content, which modulate the rate of cellular dehydration in vivo, (3) to determine the potassium efflux pathways which leads to in vivo dehydration of "fast track" cells, and (4) to determine whether sickling is a requirement for dense cell formation in vivo. A large amount of information concerning the in vitro properties of density-defined sickle cells is available, but these data are difficult to interpret due to the age obtained, using radioisotopic methods which would not be possible today. The proposed studies utilize two new non-isotopic techniques for studying age-and density-defined sickle RBC. The first takes advantage of the presence of transferrin receptors (TfR) on newly emergent sickle reticulocytes. This marker makes it possible to study a very young, age- matched population of cells in each density fraction. Extensive studies will compare the potassium flux pathways of light and heavy TfR+cells in order to determine which of several candidate pathways may be responsible for "fast track" cells. Furthermore, TfR+cells will be isolated from density-defined fractions with an immunomagnetic technique directed against TfR, and their Hb F content measured. The second novel technique is the reinfusion of a small volume of biotinylated, reticulocyte-rich light RBC. The biotin on their surface allows subsequent quantitation by flow cytometry and isolation from the circulation with streptavidin- coated magnetic beads. These cells will be used to follow time-dependent in vivo changes, including the rates of formation and removal of dense cells.